title: A mass spectrometric analysis of 4-hydroxy-2-(E)-nonenal modification of cytochrome c.
abstract: Cytochrome c is a key mitochondrial respiratory protein that is particularly susceptible to modification during oxidative stress. The nature of this susceptibility is linked to the mitochondrial membrane being rich in esterified linoleic acid, which predisposes this organelle to the formation of lipid peroxidation products such as 4-hydroxy-2-(E)-nonenal (4-HNE). To better understand the nature of cytochrome c modification by 4-HNE, we initiated an in vitro study utilizing a combination of MALDI-TOF mass spectrometry, LC-ESI-MS/MS and isotope labeling to monitor 4-HNE modification of cytochrome c under various conditions. The overwhelming reaction observed is Michael addition by Lys side-chains in addition to the modification of His 33. While the Lys-4-HNE adducts were generally observed to be reversible, the 4-HNE-His 33 was observed to be stable with half of the formed adduct surviving the denaturation and proteolysis protocols used to generate proteolytic peptides for LC-ESI-MS/MS.
authors.affiliation: Department of Chemistry, Case Western Reserve University, Cleveland, OH 44106, USA.
authors.forename: Xiaoxia
authors.initials: X
authors.lastname: Tang
authors.forename: Lawrence M
authors.initials: LM
authors.lastname: Sayre
authors.forename: Gregory P
authors.initials: GP
authors.lastname: Tochtrop
mesh_terms: D000447:Aldehydes / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods*; D045304:Cytochromes c / Q000737:chemistry* / Q000378:metabolism; D006736:Horses; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D055550:Protein Stability; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry; D014357:Trypsin / Q000378:metabolism
chemical_list: D000447:Aldehydes; D010446:Peptide Fragments; D045304:Cytochromes c; D014357:Trypsin; C027576:4-hydroxy-2-nonenal